Methods and compositions for prevention of angioproliferation

ABSTRACT

The invention provides pharmaceutical compositions comprising  Porphyromonas gingivalis  protease and hemagglutinin polypeptides that have anti-angiogenic activity and methods for their use.

PRIORITY

This application claims priority to U.S. Ser. No. 09/849,115, filed onMay 5, 2001, which is incorporated by reference in its entirety herein

FIELD OF THE INVENTION

The invention provides compositions and methods for the treatment,prevention and amelioration of angioproliferative conditions.

BACKGROUND INFORMATION

Cancer is the second leading cause of death in the United States,accounting for over one half million deaths per year. (National VitalStatistics Report, 1998, Vol. 48, No. 11). The total economic costassociated with cancer has been estimated to be over $100 billiondollars annually. (Brown, M. L. et al. In Cancer Epidemiology andPrevention, 1996). There is currently no cure for the disease, butseveral lines of research appear promising. Of these, research directedat preventing angiogenesis offers the most hope.

Angiogenesis involves a complex biochemical cascade of events that leadsto new blood vessel formation in, for example, developing tumors. Forcells to survive, each must have some communication, direct or indirectwith the existing vasculature in order to obtain nutrients and oxygen,and to offload metabolic waste products. Active vascularization isnormally observed following injury to a tissue, during development, orin response to ovulation in females. However, abnormal rapidproliferation of blood vessels is also observed in areas where cancerousmasses have developed. Because the rate of cell mass growth is limitedby the degree of vascularization present, cancer cells release chemicalsubstances into the surrounding environment to induce nearby, nourishingblood vessels to grow toward the proliferating cancer cell mass. Forexample, angiopoietin-1 basic fibroblast growth factor (bFGF), vascularendothelial growth factor (VEGF) and other substances are released fromcancer cells to coax surrounding blood vessels to grow collateralvessels toward the tumor. Once vascularized, a tumor mass may growlocally, or may metastasize and begin to spread through the bloodstreamand lymphatic system to other parts of the body, causing significantdamage. For example, vascularization of the retina in diabeticretinopathy can lead to retinal detachment resulting in blindness.Metastasis is a hallmark for malignancy, which in extreme cases may leadto rapid death of an individual. Ironically, some tumors may alsosecrete angiostatic substances to inhibit tumor growth (Chen et al.,Cancer Res. 1995, 55, 4230-4233; O'Reilly, Cell 1997, Jan. 24(88):277-285). Thus, it appears that in healthy individuals angiogenesisassociated with tumor growth may be regulated by a fine balance betweenthe release of angiogenic factors and the release of angiostaticfactors. It is believed that by blocking the process of angiogenesis,tumor growth can be suspended, which in turn would lead to cancerremission.

Most early research directed at preventing angiogenesis involvedexposing various cell lines to angiostatic compounds and assessing thedegree of proliferation either in vivo or in vitro. The National CancerInstitute, for example, uses proliferation, migration and cord formationassays in HUVE cells for its anti-angiogenesis testing. Severalangiostatic agents that function to prevent the proliferation of cancercells have been isolated and tested. For example, administration ofAngiostatin has been shown to suppress vascular endothelial cellproliferation, thereby reducing the size and lethality of tumors (FolmanJ., Forum Genova 1999 July-Dec. 9 (3 Suppl. 3): 5962). RecombinantEndostatin (baculovirus) has been used to inhibit the proliferation ofbovine capillary endothelial cells. (O'Reilly, et al., Cell, 1994, Oct.21; 79(2):185-8). Until recently, angiostatic compounds have includedonly those substances capable of preventing proliferation of cells.However, a growing body of evidence demonstrates that agents whichinhibit proliferation via cellular detachment from tumor masses performan analogous function.

Recent studies have been directed at interfering or disrupting themechanisms involved in cell-cell or cell-matrix binding as a means toreduce or eliminate cancerous growth. Cells will not proliferate if theyare not first attached to a surface. For example, impaired cell-matrixcontact leads to anoikis (epithelial apoptosis) (Vitale et al., FEBSLett. 1999 462 (1-2:57-60); Attwell et al., Oncogene 2000 19(33):3811-5;Rosen, J. Cell. Biol 2000 149(2):447-56); Rytomaa et al., Curr. Biol.1999 9(18):1043-6 or endothelial apoptosis (Erdreich-Epstein, CancerRes. 2000 Feb. 1; 60(3):71221). This anchorage dependence is mediated,in part, by cell surface molecules known as integrins. See e.g.,Erdreich-Epstein et al., Cancer Res., 2000 Feb. 1; 60(3):712-21; Lee &Juliano, Mol. Biol. Cell, 2000 Jun. 11(6):1973-87; Kawahara, J. CancerRes. Clin. Oncol, 1995 1212 (3):133-40; Lee, Mol. Biol. Cell 200011(6):1973-87; Ruoslahti, Kidney Int. 1997 51(5):1413-7; Brassard etal., Exp. Cell. Res. 1999 251 (1):33-45; Kottke et al., J. Biol. Chem.1999 274(22):15927-36.

Anti-angiogenic approaches are the most recent and promising avenue incancer treatment. Agents capable of blocking vascularization ofneoplastic tissue can prevent subsequent growth of transformed tissueand can lead to existing tissue remission. Anti-angiogenic activity hasbeen detected for several endogenous factors. For example, combrestatinA-4 disodium phosphate (CA4DP) (Dark, Cancer Res. 1997 May 15; 57(10):1829-34), a purified human PSA compound (WIPO PCT publication No.WO 99/60984), Endostatin (add, et al., Biochem. Biophys. Res. Commun.1999 Sep. 24:263(2):3405), so (AIbini, Faseb J. 1999 13(6):647-55),Epidermal Growth Factors (EGF) (Kotke, et al., J. Bio. Cam 1999 May28;274(22):15927-36), and angiostatin (Stack, Biochem J., 1999 340(pt1):77-84) have anti-angiogenic activity.

For many years, scientists have been in search of therapeutics that canbe used to prevent periodontal diseases, including gum infections andtooth decay. One organism that has been identified as a potentialetiologic agent of such pathologies as gingivitis and periodontaldisease is the pathogen Porphyromonas gingivalis. Sequences from thepathogen have been cloned and sequenced. Examples of such work can befound in U.S. Pat. Nos. 5,824,791 and 5,830,710, both of which arehereby incorporated herein by reference in their entity.

Proteolytic enzymes of Porphyromonas gingivalis are the main tool forproviding nutrients to these asaccharolytic bacteria and are alsoimportant virulence factors. These enzymes have been shown to degradebasement membrane matrix proteins (Uitto, Oral Microbiology andImmunology 1988 3:97-102), (Smalley, Arch. Oral Biol. 1988 33 (5):323-9)and purified P. gingivalis cysteine protease has been shown to disruptthe basement membrane of human carcinoma monolayer (Shah, J.Periodontol. 1992 63(1):44-51).

Targeted disruption of fibronectin-integrin interactions in humangingival fibroblasts has been demonstrated by the RI protease of P.gingivalis W50 (Scragg, et al., Infect. Immun. 1999 67(4):1837-43).Other studies have examined the function of P. gingivalis extract oncell-cell and cell-matrix bonds. Cell detachment from each other andfrom the underlying surface correlates with the cysteine-dependentproteolytic activity of P. gingivalis (Johansson, Eur. J. Oral. Sci.1998 106(4):863-71). B1-integrin, occluding and E-cadherin are targetedby P. gingivalis proteolytic activity in canine epithelial cells (Katz,et al., Infect. Immun. 2000 68(3):1441-9).

SUMMARY OF THE INVENTION

It is an object of the invention to provide compositions and methods fortreatment, prevention or amelioration of angioproliferitive conditions.This and other objects of the invention are provided by one or more ofthe embodiments described below.

One embodiment of the invention provides a composition for the t ent,prevention, or amelioration of an angioproliferitive condition. Thecomposition comprises a pharmaceutically effective amount of asubstantially purified Porphyromonas gingivalis arginine specificcysteine protease polypeptide, Porphyromonas gingivalis lysine specificcysteine protease polypeptide, Porphyromonas gingivalis hemagglutininpolypeptide, or a combination thereof, wherein the polypeptide hasanti-angiogenic activity, and a pharmaceutically acceptable excipient.The polypeptide can be selected from the group consisting of rgpA, rgpB,kgp, hag, prtT and tla polypeptides. The polypeptide can have at leastabout 90% sequence identity with a rgpA, rgpB, kgp, hag, prtT, or tlapolypeptide and has anti-angiogenic activity. The polypeptide can be abiologically functional homolog, or isoform of a rgpA, rgpB, kgp, hag,prtT or tla polypeptide. The polypeptide can be a fragment of aPorphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cystine proteasepolypeptide, or Porphyromonas gingivalis hemagglutinin polypeptide, suchas HA2. Such a fragment has anti-angiogenic activity.

An angioproliferative condition can be carcinoma, sarcoma, melanoma,benign tumor, ocular retinopathy, retrolental fibroplasias, psoriasis,angiofibromas, endometriosis, hemangioma, rheumatoid arthritis, OslerWebber Syndrome, myocardial angiogenesis, telangiectasia, hemophiliacjoints, wound granulation, intestinal adhesions, post-surgery adhesions,arteriosclerosis, scleroderma, hypertrophic scars, cat scratch disease,Helicobacter pylori ulcers, capillary proliferation withinatherosclerotic plaque, or a combination thereof.

Another embodiment of the invention provides a method for the treatment,prevention, or amelioration of an angioproliferative condition. Themethod comprises administering a pharmaceutically effective amount of aPorphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cysteine proteasepolypeptide, Porphyromonas gingivalis hemagglutinin polypeptide, or acombination thereof to a patient in need thereof, wherein thepolypeptide has anti-angiogenic activity, whereby the angioproliferativecondition is treated, prevented or ameliorated. The method can comprisecontacting a vasculature supplying a biological structure affected bythe angioproliferative condition with the polypeptide. The polypeptidecan be contacted with a basolateral surface of the vasculature.

Yet another embodiment of the invention provides a method forpotentiating effects of a chemotherapeutically effective agent. Themethod comprises administering a Porphyromonas gingivalis argininespecific cysteine protease polypeptide, Porphyromonas gingivalis lysinespecific cysteine protease polypeptide, Porphyromonas gingivalishemagglutinin polypeptide, or a combination thereof, wherein thepolypeptide has disrupts cell-cell adhesion, cell-matrix adhesion, orboth; and a chemotherapeutically effective agent to a patient. Effectsof the chemotherapeutically effective agent arm potentiated.

Still another embodiment of the invention provides a method forpreventing the formation of now vasculature required for implantation orsustenance of a fertilized mammalian ovum. The method comprisesadministering a Porphyromonas gingivalis amine specific cysteineprotease polypeptide, Porphyromonas gingivalis lysine specific cysteineprotease polypeptide, Porphyromonas gingivalis hemagglutininpolypeptide, or a combination thereof to a mammal, wherein thepolypeptide has anti-angiogenic activity, whereby the formation of newvasculature required for implantation or sustenance of a fertilizedmammalian ovum is prevented.

Even another embodiment of the invention provides a pharmaceuticalcomposition for facilitating passage of compounds through a blood-brainbarrier comprising a pharmaceutically effective amount of asubstantially purified Porphyromonas gingivalis arginine specificcysteine protease polypeptide, Porphyromonas gingivalis lysine specificcysteine protease polypeptide, a Porphyromonas gingivalis hemagglutininpolypeptide, or a combination thereof, and a pharmaceutically acceptableexcipient. The pharmaceutical composition can further include a compoundto be passed through the blood-brain barrier.

Another embodiment of the invention provides a method of delivering acompound through a blood-brain barrier of a patient comprisingadministering a pharmaceutically effective amount of a Porphyromonasgingivalis arginine specific cysteine protease polypeptide,Porphyromonas gingivalis lysine specific cysteine protease polypeptide,Porphyromonas gingivalis hemagglutinin polypeptide, or a combinationthereof and the compound, whereby the compound is passed through theblood-brain barrier.

Still another embodiment of the invention provides a method of degradinga tumor. The method comprises administering a pharmaceutically effectiveamount of a Porphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cysteine proteasepolypeptide, Porphyromonas gingivalis hemagglutinin polypeptide, or acombination thereof to a patient having a tumor, wherein the polypeptidedegrades cell-cell bonds, cell-matrix bonds, or both cell-cell bonds andcell-matrix bonds of the tumor, whereby the tumor is degraded.

Therefore, the present invention discloses anti-angiogenic compositionsof mater and methods of their use, which are capable of disruptingendothelial tissue growth and proliferation. The methods involve localor systemic application of a P. gingivalis polypeptide to a targetedtissue. The disclosed polypeptides can inhibit angiogenesis associatedwith malignant tumor proliferation by, for example, disruptingendothelial layer cell-cell and cell-matrix adhesion bonds. Use of thesecompounds and methods have advantages over conventional treatments suchas chemotherapy, because it targets only growing vessels, while leavingintact vessels unaffected.

The present invention provides a novel strategy that uses protein,peptide and nucleic acid sequences of Porphyromonas gingivalis to treator prevent angioproliferative conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates percent detachment of active Human Umbilical VeinEndothelial Cells (HUVEC) after 24 hours of treatment with a proteinextract from P. gingivalis. Represented are mean values from triplicateexperiments.

FIG. 2 demonstrates percent detachment of active HUVEC after 48 hours oftreatment with a protein extract from P. gingivalis.

FIG. 3 demonstrates percent detachment of quiescent HUVEC after 24 hoursof treatment with a protein extract from P. gingivalis.

FIG. 4 demonstrates percent detachment of quiescent HUVEC after 48 hoursof treatment with a protein extract from P. gingivalis.

FIG. 5 demonstrates percent detachment of active human non-small celllung carcinoma cell line (A549) after 24 hous of treatment with aprotein extract from P. gingivalis.

FIG. 6 demonstrates percent detachment of active human non-small celllung carcinoma cell line (A549) after 48 hours of treatment with proteinextract from P. gingivalis.

FIG. 7 demonstrates percent detachment of quiescent human non-small celllung carcinoma cell line (A549) after 24 hours of treatment with aprotein extract from P. gingivalis.

FIG. 8 demonstrates percent detachment of quiescent human non-small celllung carcinoma cell line (A549) after 48 hours of treatment with aprotein extract from P. gingivalis.

FIG. 9 demonstrates the reduction in human vascular endothelial cellmigration after exposure to a protein extract from P. gingivalis over a24 hour period.

FIG. 10 demonstrates detachment of human non-small cell lung carcinomacell line (A549) after treatment with a proteinase extract of P.gingivalis alone and with an extract of Bacteroides fragilis expressingPrtP protease from P. gingivalis.

FIG. 11 is a table showing the degree of proliferation inhibition of aHUH7 cell line exposed to P. gingivalis and E. coli extract, and to P.gingivalis cells in the presence or absence of inhibitors.

FIG. 12A-E shows the results of reactivity of a HUH7 cell line withanti-occludin antibodies. FIG. 12A depicts non-treated control HUH7cells. FIG. 12B depicts the HUH7 cells treated with P. gingivalisextract. FIG. 12C depicts HUH7 cells treated with P. gingivalis extractin the presence of the inhibitor TLCK. FIG. 12D depicts results oftreatment with heat-treated P. gingivalis extract. FIG. 12E depicts HUH7cells treated with a control E. coli extract; the occludin network isintact.

FIG. 13A-D show the result of reactivity of a HUH7 cell tine with ananti-pan cadherin antibody. FIG. 13A depicts non-treated control HUH7cells. FIG. 13B depicts HUH7 cells treated with P. gingivalis extract.FIG. 13C depicts HUH7 cells treated with P. gingivalis extract in thepresence of inhibitor TLCK. FIG. 13D depicts HUH7 cells treated withheat inactivated P. gingivalis extract.

FIG. 14A-C shows proliferation inhibition of a HUVEC polarized cellline. FIG. 14A depicts non-treated, control polarized human endothelialcell layer, ECV-304. FIG. 14B shows ECV-304 cells treated basolaterally(lower chamber) with a 60% fraction of P. gingivalis culture liquidproteins. FIG. 14C depicts the results of ECV-304 cells lumenallytreated with a 60% fraction of P. gingivalis culture liquid proteins.

FIG. 15 demonstrates total cell number reduction as result of treatmentwith P. gingivalis strain W83 extract.

FIG. 16 demonstrates that detachment of HUVE cells is reduced byinhibitors or heating of SPF.

FIG. 17 demonstrates transendothelial resistance of HUVE cells upontreatment with P. gingivalis soluble protein fraction (SPF) over aperiod of 6 hours.

FIG. 18 demonstrates transendothelial resistance of HUVE cells upontreatment with P. gingivalis soluble protein fraction (SPF) over aperiod of 8 days.

FIG. 19A-C shows contrast micrographs of HUVE cell layers that weretreated with P. gingivalis SPF or mock-treated at day 8. FIG. 19A showstreated cells. FIG. 19B shows BL-treated cells. FIG. 19C showsmock-treated control cells.

FIG. 20A-C show contrast micrographs of HUVE cells treated with extractsof E. coli. FIG. 20A shows HUVE cells treated with control E. coli hostextract. FIG. 20B shows control mock-treated cells. FIG. 20C shows cellstreated with extracts of E. coli host cells that express a 15 kDainternal fragment of a P. gingivalis HagA repeat.

FIG. 21 shows proliferation inhibition of HUVE cells treated with HA215-kDa P. gingivalis protein cloned in E. coli. (E. coli 0.5: control E.coli protein extract at 0.5 mg/ml. E. coli HA2 0.5: E. coli proteinextract containing HA2, the 15-kDa polypeptide at 0.5 mg/ml.

FIG. 22 shows potential pathways for leakage from blood vessels intumors. Intercellular openings (arrows) between lining cells of a murinetumor vessel viewed by scanning EM. FIGS. 22A and B: Multiple largeintercellular openings (arrow, FIG. 22A) and three smaller transcellularholes (arrows, FIG. 22B) in branched lining cells of a tumor vessel. Theintercellular openings are much larger than the holes. The boxed regionin FIG. 22A is shown at higher magnification in FIG. 22B.

DETAILED DESCRIPTION OF THE INVENTION

“Angiogenesis” or “angioproliferation” means conditions of rapiddevelopment of vascular supply to a particular organ or biological site.The rapid development can be uncontrolled and/or pathogenic, as in thedevelopment of a tumor. However, angiogenisis or angioproliferation canalso be associated with non-pathological conditions such as angiogenesiswhich occurs upon implantation of a fertilized ova. An anti-angiogenicor anti-angioproliferative composition is capable of reducing orpreventing angiogenisis or angioproliferation.

The present invention is directed to arginine- or lysine-specificcysteine protease polypeptides and hemagglutinin polypeptides derivedfrom P. gingivalis. The polypeptides can be used to treat or preventangioproliferative conditions, including but not limited to melanoma,sarcoma, and carcinomas including, for example, breast, colon, lung andprostate carcinomas. Additional pathologies susceptible to treatmentaccording to the present invention include benign tumor, ocularretinopathy, retrolental fibroplasias, psoriasis, angiofibromas,endometriosis, hemangioma, rheumatoid arthritis, Osler Webber Syndrome,myocardial angiogenesis, telangiectasia, hemophiliac joints, woundgranulation, intestinal adhesions, post-surgery adhesions, scleroderma,hypertrophic scars, cat scratch disease, and Helicobacter pylori ulcerand capillary proliferation within atherosclerotic plaque. Post-surgeryadhesions are common complication of gynecologic and abdominal surgery.Such complications can lead to infertility, ectopic pregnancy, chronicpain, prolonged recovery and intestinal obstruction. Adhesions followingsurgery can be prevented by applying polypeptides of the invention atthe end of the operation.

Furthermore, the compositions of the invention can be used to controlnon-pathogenic angiogenic conditions, such as using the compositions asa contraceptive to prevent implantation of fertilized ova.

Polypeptides of the Invention

Compositions of the invention comprise a substantially purifiedPorphymonas gingivalis arginine-specific cysteine protease polypeptide,a Porphymonas gingivalis lysine-specific cysteine protease polypeptide,a Porphymonas gingivalis hemagglutinin polypeptide or combinationsthereof. These polypeptides are expressed from members of the P.gingivalis protease and hemagglutinin gene family. See e.g., Curtis etal., J. Periodont. Res. 34:494 (1999). “Substantially purified” refersto polypeptides that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

Polypeptides of the invention include rgpA arginine-specific cysteineproteases. P. gingivalis rgpA proteases are a group of proteasesproduced by genes of a homologous loci in different of P. gingivalis.Examples of the rgpA proteases are: prpR1 (Accession number X82680),rgp-1 (Accession number U15282), rgpA (Accession number D26470), prtR(Accession number L26341), prtH (Accession number L27483), and hagE(Accession number AF026946), which is similar to rgp (Accession numberA55426). Isoforms of rgpA proteases are also part of the invention andinclude, for example, RgpA_((cat)), mt-RgpA_((cat)) and HRgpA isoforms.See, e.g., Curtis et al., J. Periodont. Res. 34:494 (1999).

P. gingivalis rgpB arginine-specific proteases are also polypeptides ofthe invention Examples of rgpB proteases include prtRII (Accessionnumber AF007124), rgp-2 (Accession number U85038), rgpB (Accessionnumber D64081), and prR2 (Rangarajan, Mol Microbiol 1997 Mar.23(5):955-65). The rgpB proteases are all highly related and are derivedfrom homologous genes which occur in different strains of P. gingivalis.Isoforms of rgpB proteases are also part of the invention and include,for example, RgpB and mt-RgpB isoforms. See e.g., Curtis et al., J.Periodont. Res. 34:494 (1999).

Lysine-specific proteases of P. gingivalis include kgp proteases. kgpproteases include, for example, prtK (Accession number U75366), kgp(Accession numbers U54691; D83258), prtP (Accession numbers U42210;AF017059), kgp(381)-hagD (Accession number U68468). The kgp proteasesare all highly related and are derived from homologous genes indifferent stains of P. gingivalis. Isoforms of kgp proteases are alsoincluded in the invention.

Hemagglutinin polypeptides of the invention include hagA, hagB, hagC,hagD, hagE, tla (e.g., Accession number Y07618) and prtT (Accessionnumber S75942; M83096). See also, e.g., U.S. Pat. Nos. 5,824,791 and5,830,710 and Aduse-Opoku et al., J. Bacteriol. 179:4778-4788 (1997). Asdisclosed in U.S. Pat. No. 5,824,791, a hag gene can be a 7887 bpmolecule, which encodes a gene product of 2628 amino acids. Within thegene product, there are four repeat segments: HArep1, HArep2, HArep3 andHArep4. Bach of these segments contains a 15-kDa fragment, HA2, thatpossesses anti-angiogenic activity and is found in the secreted proteinfraction. HA2 is instrumental in production of necrosis, which leads toperiodontal disease. HA2 is located within a hagA sequence between aminoacids 683 and 819 in the first repeat and is similarly located in theother repeats (see Han et al., Infect. Immun. Page 4002, FIG. 2, U.S.Pat. Nos. 5,824,791 and 5,830,710). P. gingivalis hemagglutininpolypeptides have cysteine protease activity against arginine containingsubstrates. Nishikata and Yoshimura, Biochem Bio. Phys. Res. Comm178:336-40 (1991). P. gingivalis hemagglutinins also function asattachment factors and the substrate binding site is responsible forattachment to erythrocytes. Hemagglutinins also contain “peptidase C25family” activity. The entire hagA, hagB, hagC, hagD, hagE, prtT or tlapolypeptide can be used to produce anti-angiogenic effects.Alternatively, any of the HArep sequences or HA2 can be usedAdditionally, compounds with similar activity can also be developedwhich have equal or greater potency than polypeptides of the invention,and such compounds come within the scope of this invention. For example,one of skill in the art can design peptidomimetics using, for example,directed protein evolution or by minimizing the size of the polypeptideso that improved or smaller versions of the polypeptide are developed.

Polypeptides of the invention include polypeptides produced from any P.gingivalis rgpA, rgpB, kgp, hag, prtT or tla gene. It is well known inthe art that the proteases and hemagglutinins of P. gingivalis areproduced from a family of protease and hemagglutinin genes that arehighly related. This gene family is known to vary from strain to strainof P. gingivalis. See e.g., Curtis et al., J. Periodont. Res. 34:494(1999). The invention includes all polypeptides produced from the rgpA,rgpB, kgp, hag, prtT or tla gene family that have anti-angiogenicactivity. One of skill in the art could determine whether a protease orhemagglutinin gene or polypeptide fell within the rgpA, rgpB, kgp, hag,prtT or tla family by comparing the nucleotide or protein sequence ofthe gene or protein in question to the known sequences of these genesand proteins.

Curtis et al. (J. Peridont. Res. 34:464 (1999)) describes these genesfamilies and discloses examples of genes that fall within the P.gingivalis protease and hemagglutinin gene family. U.S. Pat. Nos.5,824,791 and 5,830,710 further describes genes that fall within the hagfamily. All of these related polypeptides have cysteine proteaseactivity and sequence homology and as such, can be classified into theprotease-hemagglutinin family.

Polypeptides of the invention can either be full-length polypeptides orfragments of polypeptides. For example, fragments of polypeptides of theinvention can comprise about 10, 25, 50, 100, 200, 250, 500, 750, or1,000 amino acids of polypeptides of the invention Percent sequenceidentity ha an art recognized meaning and there are a no of methods tomeasure identity between two polypeptide or polynucleotide sequences.See, e.g., Lesk, Ed, Computational Molecule Biology, Oxford UniversityPress, New York, (1988); Smith, Ed, Biocomputing: Informatics And GenomeProjects, Academic Press, New York, (1993); Griffin & Griffin, Eds.,Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey,(1994); von Heinje, Sequence Analysis In Molecular Biology, AcademicPress, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer,M Stockton Press, New York, (1991). Methods for aligning polynucleotidesor polypeptides are codified in computer programs, including the GCGprogram package (Devereux et al., Nuc. Acids Res. 12:387 (1984)),BLASTP, BLASTN, FASTA (Atschul et al., J. Mole Biol. 215:403 (1990)),and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) which uses the local homology algorithm ofSmith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, thecomputer program ALIGN which employs the FASTA algorithm can be used,with an affine gap search with a gap open penalty of −12 and a gapextension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Amino acid sequences having at least about 75, preferably at least about90, 95, 96, 98, or 99% sequence identity to amino acid sequences of P.gingivalis rgpA, rgpB, kgp, hag, prtT or tla polypeptides and haveanti-angiogenic activity are also polypeptides of the invention.

Also included in the invention are polypeptides having amino acidsequences which are at least about 75, preferably at least about 90, 95,96, 98, or 99% sequence identity toamino acid sequences of P. gingivalisrgpA, rgpB, kgp, hag, prtT or tla proteins that are produced by anorganism other than P. gingivalis and have anti-angiogenic activity.

Polypeptides of the invention also are biologically functional homologs,analogs and isoforms of rgpA, rgpB, kgp, hag, prtT or tla polypeptides.A biologically functional homolog is a polypeptide that has sequenceidentity to an analogous polypeptide from another species. Homologs canbe naturally occurring or can be a polypeptide that does not occur innature. An isoform is encoded by a distinct mRNA splice variant and canbe a naturally occurring polypeptide or can be a polypeptide that doesnot occur in nature. An analog is a polypeptide having alterationsinvolving one or more amino acid insertions and deletions and/orconservative amino acid substitutions. A homolog, analog or isoform ofthe invention comprises at least about 75, 90, 95, 96, 98 or 99%sequence identity to a rgpA, rgpB, kgp, hag, prtT or tla polypeptide. Abiologically functional homolog, analog or isoform has at least onebiological activity of a rgpA, rgpB, kgp, hag, prtT or tla polypeptide.That is, they are capable of preventing endothelial cell proliferation,disruption of vascular endothelium, promoting cellular detachment,inhibiting migration of endothelial cells, blocking formation of newblood vessels, or destroying existing blood vessels feeding, forexample, tumors.

A biologically functional homolog or analog, isoform, or fragment can betested for biological activity using, for example a cellproliferation/cell detachment assay (see Example 1), a migrationinhibition assay (see Example 2), an occludin-stain junction assay (seeExample 7), catherin-stain junction assay (see Example 8) proliferationinhibition of polarized cell assay (see Example 10), degradation of α5β1integrin assay (see Example 11), or transendothelial resistance assay(see Example 12). A biologically functional homolog, analog, isoform orfragment has about 85%, 90%, 95%, 98%, 99%, 100%, 105%, or 110%biological activity of a specific specie in question.

In one embodiment of the invention, a polypeptide of the invention isproduced recombinantly. A polynucleotide encoding a polypeptide of theinvention can be introduced into a recombinant expression vector, whichcan be expressed in a suitable expression host cell system usingtechniques well known in the art. A variety of bacterial, yeast, plant,mammalian, and insect expression systems are available in the art andany such expression system can be used. Optionally, a polynucleotideencoding a polypeptide can be translated in a cell-free translationsystem. A polypeptide of the invention can also be chemicallysynthesized.

If desired, a polypeptide can be produced as a fusion protein, which canalso contain other amino acid sequences, such as amino acid linkers orsignal sequences, as well as ligands useful in protein purification,such as glutathione-S-transferase, histidine tag, and staphylococcalprotein A. More than one polypeptide of the invention can be present ina fusion protein.

Biological Activity of Polypeptides of the Invention

Polypeptides of the invention have anti-angiogenic biological activity.That is, they are capable of preventing endothelial cell proliferation,disruption of vascular endothelium, promoting cellular detachment,inhibiting migration of endothelial cells, blocking formation of newblood vessels, or destroying existing blood vessels feeding, forexample, tumors or combinations thereof. Therefore, compositions of theinvention are capable of inhibiting of tumor enlargement, decreasingtumor mass, and inhabiting, reducing, or preventing related biologicalprocesses associated with angiogenesis and vascular supply to aparticular biological organ or location, or combinations thereof. Inaddition, the polypeptides of the invention can be capable ofdisintegrating cell-to-matrix and cell-to-cell bonds in, for example, atumor such that the tumor is reduced or eliminated. A polypeptide of theinvention has at least one of the above-mentioned biological activities.

Polypeptides of the invention can act on cell surface adhesion molecules(CAM's), from the basolateral side of the endothelium. Polypeptides ofthe invention can inhibit angiogenesis by targeting integrin incell-cell and cell-matrix adhesion bonds. Of particular importance isthe fact that vasculature supplying tumor tissues is aberrantly leaky.Regular blood vessels are well formed and have a well-developed adhesionsystem to keep them together, while blood vessels supplying tumors arepoorly formed and extremely leaky (Herlyn, Immunother. 1999 May,22(3):185); see also, Hashizume, et al., Am. J. Pathol. 2000 April156(4):1363-80). As a result, locally or systemically administeredpolypeptides of the invention leak out of the vasculature that suppliestumor tissue. Access to the basolateral surface of the vasculature isthereby achieved, which results in disruption of the vascular at thatlocation such that di-ation of the tumor occurs. Therefore, compositionsof the invention can disrupt angiogenesis without affecting integrity ofnormal blood vessels.

Methods of Treatment

Compositions of the invention can be used to treat several conditions inmammals, including humans. For example, angioproliferative conditionscan be treated, prevented or ameliorated according the invention.Methods of treatment comprise administering to a patient having anangioproliferative condition a pharmaceutically effective mount of acomposition of the invention such that an anti-angiogenic effect isachieved Angioproliferative conditions include, for example, carcinoma,sarcoma, melanoma, benign tumor, ocular retinopathy, retrolentalfibroplasias, psoriasis, angiofibromas, endometriosis, hemangioma,rheumatoid arthritis, Osler Webber Syndrome, myocardial angiogenesis,telangiectasia, hemophiliac joints, wound granulation, intestinaladhesions, post-surgery adhesions, atherosclerosis, scleroderma,hypertrophic scars, cat scratch disease, Helicobacter pylori ulcers,capillary proliferation within atherosclerotic plaque, or a combinationthereof.

An angioproliferative condition can be selectively treated according tothe invention by contacting vasculature supplying a biological structureaffected by an angioproliferative condition with an anti-angiogenicallyeffective amount of a composition of the invention. Due to the knownleakiness of vasculature supplying, for example, a tumor, local orsystemic administration of a composition of this invention facilitatescontact with the basolateral surface of said vasculature, including theendothelium, with no effect on normal tissues.

Compositions of the invention can also be used to potentiate the effectsof a chemotherapeutically effective agent. Such methods compriseco-administering a chemotherapeutically effective agent in the presenceof a polypeptide of the invention effective to disrupt cell-celladhesion, cell-matrix adhesion, or both. Such co-administration can bein the form of a covalent complex, an ionic complex, a mixturesimultaneous but separate administration, or administration within arelatively close temp sequence. Appropriate chemotherapeutic agentsinclude, but are not limited to doxorubicin, daunorubicin, doxorubicin,idarubicin, vincristine, 6-mercaptopurine, 6-thioguanine, methotrexate,cytoxan, cytarabine, L-asparaginase, busulfan, cyclophoshamide,melphalan, carmustine, lomustine, 5-fluorouracil methotrexate,fludarabine, bleomycin, docetaxel etoposide, vinorelbine, antibodies,and the like.

Polypeptides of the invention can also be used in contraception methods.During pregnancy, the endometrial layer of the uterus becomes thickenedand engorged with blood vessels upon implantation of a fertilized ovum.Without a well-developed vasculature the fertilized ovum will not besustained, and the endometrial layer will be sloughed-off in the form ofmenses, i.e., menstruation. In one embodiment of the present invention,therapeutic compositions for use as contraceptives are provided. Inorder to induce contraception, the internal vasulature of the uterus iscontacted with a contraceptively effective amount of a polypeptide ofthe invention. The mode of achieving bioavailability of polypeptides ofthe invention in this and other angioproliferative conditions can be,for example, through systemic or localized administration, such asintrauterine infusion.

Polypeptides of the invention can also be used to facilitate passage ofcompounds, such as pharmaceuticals, through the blood-brain barer of amammal. A polypeptide of the invention can be administered along with acompound to permeablize a blood-brain barrier and allow delivery of thecompound thought the blood-brain barrier. Such methods compriseco-administering a compound in the presence of a polypeptide of theinvention effective to disrupt cell-cell adhesion, cell-matrix adhesion,or both. Such co-administration can be in the form of a covalentcomplex, an ionic complex, a mixture, simultaneous but separateadministration, or administration within a relatively close temporalsequence.

Pharmaceutical Compositions and Administration Thereof

A pharmaceutically effective amount refers to an amount effective intreating an angiogenic condition in a mammalian patient, such as ahuman. A pharmaceutically acceptable excipient is a non-toxic carrier,vehicle or adjuvant that can be administered to a patient, together witha polypeptide of this invention, and which does not destroy thepharmacological activity of the polypeptide. Excipients, includingcarriers, vehicles and adjuvants are well known in the art See, e.g.,Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co.,Easton Pa., 1985. A pharmaceutically effective amount is an amount of apolypeptide that achieves a specified functional result. A dosage of apolypeptide of the invention can be determined using routineexperimentation Pharmaceutical compositions of the invention can beadministered by any number of routes including, but not limited to oralcontrolled release, intravenous, intramuscular, intra-arterial,intramedullary, intradermal intrathecal intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal parenteral, topical, or rectalmeans. Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, sips, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical formulations suitable for parenteral administration canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions cancontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol or dextran. Additionally,suspensions of the active compounds can be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oily or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers also can be used for delivery. Optionally, the Z ion alsocan contain suitable stabilizers or agents which increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions. For topical or nasal administration, penetrants appropriateto the particular barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

A polypeptide of the invention can also be delivered to a patient byadministration of a polynucleotide encoding a polypeptide of theinvention. Preferably, injection of a P. gingivalis protease orhemagglutinin polynucleotide, which encodes a polypeptide of theinvention, is used to treat, ameliorate, or prevent anangioproliferative condition. In addition to the practical advantages ofsimplicity of construction and modification, injection of apolynucleotide results in the synthesis of a polypeptide of theinvention in the host. The polynucleotide is preferably delivered as“naked DNA” or in a vector. A vector can be a plasmid, such as pBR322,pUC, or ColE1, or an adenovirus vector, such as an adenovirus Type 2vector or Type 5 vector. Optionally, other vectors can be used,including but not limited to Sindbis virus, simian virus 40, alphavirusvectors, poxvirus vectors, and cytomegalovirus and retroviral vectors,such as murine sarcoma virus, mouse mammary tumor virus, Moloney murineleukemia virus, and Rous sarcoma. Minichromosomes such as MC and MC1,bacteriophages, phagemids, yeast artificial chromosomes, bacterialartificial chromosomes, virus particles, virus-like particles, cosmids(plasmids into which phage lambda cos sites have been inserted) andreplicons (genetic elements that are capable of replication under theirown control in a cell). A polynucleotide can be, for example, injectedintramuscularly to a mammal at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5,5 or 10 mg/kg.

In vivo delivery of polynucleotides (e.g., plasmid DNA) into results inthe cellular uptake and expression of the polynucleotide into a desiredpolypeptide (Wolff, J. A. et al., Science 247:1465-1468 (1990); Wheeler,C. J. et al, Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)). Theefficiency of in vivo polynucleotide administration can be increasedusing, for example, chemical agents or physical manipulations. Suchchemical agents include cellular toxins such as bupivacaine, cardiotoxinor barium chloride, polymers such as polyvinyl pyrolidone, polyvinylalcohol polyethyleneimine, polyamidomine, and polyethyleneglycol-polyethyleneimine-transferrin complexes that coat the DNA andprotect it from DNases and enhance plasmid DNA-based expression orimmune responses, particles that interact with the DNA and act ascarriers and enhance DNA expression such as nanospheres, microspheres,dendrimers, collagen and polylactide co-glycolides, bulking agents suchas sucrose, detergents such as sodium glycocholate, sodium deoxycholate,and beta-cyclodextrin, cationic or non-cationic lipids, DNA bindingagents, or agents that enhance plasmid DNA transcription such as histonedeacetylase inhibitor FR901228 or S-Bromo-cyclic AMP. Physicalmanipulations include removal of nerves that control muscle contraction,electroporation, use of intravascular pressure, use of sutures coatedwith plasmid, use of sponges soaked with DNA as intramuscular depots toprolong DNA delivery, use of special needle-based injection methods, andof needleless-injectors tat propel the DNA into cells.

The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol at a pH range of 4.5 to5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can befound in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES(Maack Publishing Co., Easton, Pa.). After pharmaceutical compositionshave been prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition. Such labeling wouldinclude amount, frequency, and method of administration.

Because of the specificity of polypeptides of the invention for rapidlydeveloping vasculature, it is possible to define doses of thepolypeptides that are non-toxic to the remainder of a patient, but whichnonetheless provide a localized effect to achieve beneficialanti-angiogenic effects. Dosages of a polypeptide of the invention canbe about 0.01, 0.1, 1, 2, 5, or 10 mg/kg, of body weight, but one ofordinary skill in the art would recognize that for particularapplications, it can be necessary to use either greater or lesserdosages. If a polypeptide of the invention is found to generate animmune response in a patient, processes are known in the art formimicking biological activity of proteins through development ofminienzymes, DNA agents, or other small molecules, repeat administrationof active site mimics including for chronic administration.

The above disclosure generally describes the present invention, and allpatents, patent applications, scientific references cited in thisdisclosure are expressly incorporated herein. A more completeunderstanding can be obtained by reference to the following specificexamples which are provided for purposes of illusion only and are notintended to limit the scope of the invention.

EXAMPLE 1

Methods for Testing Human Endothelial or Carcinoma Cell DetachmentExposed to P. gingivalis Extract

To determine the efficacy of P. gingivalis protease extracts ininhibiting cell proliferation, two cell lines were tested. Proliferationinhibition was assessed by determining the detachment of tissue culturecells from their substrate. A549 human non-small cell lung carcinomacell line (maintained in RPMI-1640 medium (Gibco #11875-135)supplemented with L-Glu and Pen/Strep) and human endothelial cell line(HUVEC, ATCC #ECV-304), which was maintained in M199 medium,supplemented with L-Glutamine (0.05%, Gibco 25030-081), 0.1%Penicillin-Streptomycin (Gibco 15140-122) and 10% PBS (HyClone#SH30071.02, added after heat treatment at 56° C. for 30 min), were usedas targets in a cell detachment assay. Exponentially growing (Ex) andquiescent (Plateau) phases of cell culture growth were tested. To obtainextract for activity tests, exponentially grown broth cultures of P.gingivalis strain W83 were pelleted, resuspended in 50 mM HEPES buffer(pH 7.5), and sonicated on ice for 2 min. Following centrifugation at14,000 rpm at 4 C, the supernatant was filtered through 0.22 μmnon-protein-binding filter (Gelman) and stored at −80° C. until needed.Protein concentration was measured using Sigma bicinchoninic acidreagent (#B-9643), BSA standard solution and spectrophotometer ShimadzuUV-1201.

Six plates per sample with three plates added for non-treated controlwere used. 10⁴ cells per 60-mm tissue culture plate were used to seedthe sample. Detached cells were counted by collecting the medium aftertreatment (24 or 48 hrs), washing the attached cells with calcium-freephosphate-buffered saline (PBS) and adding the wash to the medium. Thepellet was then resuspended in medium to obtain a countable number onthe hemocytometer. The 25-square (0.1 μl volume) count ×10⁴ gives thecell count per ml. Two readings were made of each of the three plates.This number represented the detached cell count.

The attached cells were trypsinized and collected as above; the platewas washed with PBS and added to the tube. After pelleting andresuspending, two readings were made per each of the three plates toobtain the attached cell count. The mean value of the counts was taken,total number of cells (attached+detached) was obtained and thepercentage of detached cells was calculated. Different concentrations ofP. gingivalis protein extract were used in the beginning to establisheffective concentration to be used throughout. The working concentrationwas chosen to be 0.4 mg total protein tact per ml cute medium.

FIG. 1 and FIG. 2 show non-confluent (“log”) culture treated with 0.4 mgP. gingivalis extract/ml for 24 hours (FIG. 1) and 48 hours (FIG. 2).FIG. 3 and FIG. 4 show confluent culture treated for 24 hours (FIG. 3)and 48 hours (FIG. 4), respectively. FIG. 1 shows a 70% detachment foractive HUVEC culture cells following treatment with a P. gingivalisextract over a 24-hour period. FIG. 2 shows a 90% detachment of the samecells after 48 hours. FIG. 3 shows approximately 68% detachment ofquiescent Human (HUVEC after 24 hours of treatment, whereas the samecells exhibit nearly 100% detachment after 48 hours (see FIG. 4).

FIG. 5 and FIG. 6 show non-confluent (“log”) culture treated for 24hours (FIG. 5) and 48 hours (FIG. 6) with 0.4 mg P. gingivalisextract/ml medium. FIG. 7 and FIG. 8 show confluent cultures treated for24 hours (FIG. 7) and 48 hours (FIG. 8). The control is untreated A549.FIG. 5 shows active A549 human non-small cell lung carcinoma exhibited a95% detachment rate after 24 hours of treatment with a P. gingivalisextract. After 48 hours, there was no change in the percent detachmentof these same cells (see FIG. 6). FIG. 7 shows 40% detachment rate after24 hours, whereas after 48 hours the detachment rose to greater than50%. Detachment from the plastic surface of the petri dish is consistentwith the ability of P. gingivalis extract to degrade β1 integrin.Anti-angiogenic and tumor-reducing activity correlates with the degreeof detachment of the endothelial and cancer tissue culture cells in both24 and 48-hour treatments. Demonstrating detachment of up to 96% of thecells (A549) or 9% (HUVEC) can thus be considered pertinent activity.This is for total protein where the fraction of the active ingredient issmall. Therefore, its activity is very high. Both exponentially growing(“log”) and stationary phase (“plateau”) quiescent cultures showeddifferences in ability to remain attached following treatment

FIG. 15 total cell number reduction a result of the treatment. Thetreatment is more efficient on growing tumor cells than on cells instationary phase. Similar reduction was obtained with endothelial(HUVEC) cells.

FIG. 16 demonstrates that detachment of HUVEC is reduced by inhibitorsor heating of SPF (soluble protein faction; see Example 9). In order todetermine if the junctional protein-degrading activity is a cysteineproteinase, additional treatments were included Nα-p-tosyl-L-lysinechloromethyl ketone (TLCK), a cysteine proteinase inhibitor, was addedat 10 mM. In addition, heat-inactivation (20 min at 65° C.) of the P.gingivalis extract was performed to inactivate protein-mediatedproteolysis. All experiments were done in triplicate. Using a Coultercounter, the number of the detached cells was determined and mean valueswere expressed as percent of the total (detached+attached) cell number.The results of this experiment demonstrate that a P. gingivalis proteinis the active substance and that this protein is a cysteine proteinase.

EXAMPLE 2

Method of Demonstrating Detachment Associated with PrtP Protease from P.gingivalis

To demonstrate that protease PrtP isolated from P. gingivalis isresponsible for the detachment observed in Example 1, three samples wereapplied to A549 lung carcinoma cells. A single P. gingivalis protein,PrtP protease, was expressed in Bacteroides fragilis, a species relatedto P. gingivalis, but which does not express PrtP, for the purpose offurther chromatographic purification. Treatment of carcinoma cells wasperformed with an extract of B. fragilis containing PrtP and compared tothe same treatment with the wild-type B. fragilis host. Therefore, thedifference between the treatments was limited to the presence/absence ofP. gingivalis PrtP protease only.

The strains were grown in BHIS broth (per liter, 37 g Brain HeartInfusion (Difco), 1 g L-Cysteine (Sigma), 5×10⁻⁴% hemin, 0.2% NaHCO₃ inan anaerobic chamber with an atmosphere of 5% CO₂, 10% H2, and 85% N₂).Agar (1.5%) was added for solid medium. P. gingivalis W83 was grown onTrypticase soy agar (BBL Microbiology stems, Cockeysville, Md.)supplemented with sheep blood (5%), hemin (5 mg/ml), and menadione (5mg/ml). When broth-grown P. gingivalis was required, cultures were grownin Todd-Hewitt broth (BBL Microbiology Systems) supplemented with hemin(5 μg/ml), menadione (5 μg/ml), and glucose 2 mg/ml) anaerobically.Normal Bacteroides fragilis was used as a control.

Detachment of cells exposed to both B. fragilis with PrtP and P.gingivalis were nearly equal, whereas those cells exposed to the controlexhibited minimal detachment. This study provides direct evidence thatPrtP is active in cell proliferation inhibition.

A migration inhibition (“scratch wound”) assay is a method that isroutinely used for the ration of the ability of cells to migrate, animportant step in tumor neovascularization. This assay is used by theBiological Testing Branch, Developmental Therapeutics Program at NCI toidentify new anti-angiogenic compounds. Inhibition of migration isconsidered one aspect of anti-angiogenic activity (Yeh et al., MolPharmacol. 2001 May,59(5):1333-42.). FIG. 9 demonstrates a HUVECmigration inhibition assay: For this assay, endothelial cells werecultured on a slide. Upon reaching confluency, a 2-mm scrape “wound” wasintroduced on a central portion of the slide. The number of cells whichmigrated into the denuded area was enumerated after further incubationfor 24 hours. The results (the mean of 2 experiments) demonstrate thatat 0.4 mg total protein/ml, the migration of the human vascularendothelial cells was reduced by 45%.

EXAMPLE 3

Migration Inhibition Assay

To demonstrate that a P. gingivalis extract exerts anti-angiogeniceffects, as opposed to general inhibition of cell proliferation, thefollowing assay was performed. P. gingivalis extracts were produced andhomogenized to obtain an extract as described in Example 1. At 0.4 mgtotal protein/ml, human vascular endothelial cell migration in standardin vitro assay known in the at to reflect angiostatic and anti-tumoracidity, was reduced by 45%, (mean value of 2 experiments). In addition,at 48 hours, detachment of 85% of log phase lung carcinoma cells wasobserved (FIG. 10).

Since total cell protein was used, where the faction of the activeingredient is small, this experiment demonstrates that the angiostaticactivity of the P. gingivalis proteinase is high.

EXAMPLE 4

Identification of Epithelial Cell Ligands of Hemagglutinin A

In order to determine if HagA interacts directly with host cellcomponents, a functionally active fragment of HagA was produced in E.coli using the E. coli expression vector, pET19b (Novagen). In thissystem, purification was achieved by fusing a histidine tag to a Hagfragment and by affinity purification of the fusion protein on a Ni²⁺column. Oligonucleotides were designed flanking 2 HArep sequences toinclude the active site of hemagglutination as disclosed in U.S. Pat.No. 5,824,791 and to include restriction sites for ligation of thefragment into the expression vector, pET19b. The 3 kb PCR product wascloned into pT7Blue vector (Novagen), digested with NdeI and XhoL andthe coding sequence directionally subcloned into pET19b, which had beendigested with the same enzymes and CIP-treated.

Using PCR with a mixed pair of primers T7 (from vector) and ST2/3′ (frominsert), transformants in E. coli Novablue (Novagen) were screened foran insert in the proper orientation. One such clone, pEKS5, was chosenfor further work and was transformed into E. coli BL21 (DE3), anexpression strain (Novagen). After induction with 1 mM IPTG, cells werelysed and the lysate was applied to an activated His-Bind resin affinitychromatography column. Elution with 1 M imidazole-containing bufferproduced a single protein species with an apparent molecular mass of˜100 kDa. After transfer onto a nitrocellulose membrane, the protein wasprobed with anti-HagA antibody, 61BG1.3, and its authenticity wasconfirmed.

The purified recombinant HagA peptide was tested for binding to cellcomponents of two human cell lines using the Far Western Immunoblot. Forthis assay, KB oral epithelial cells and human umbilical cordendothelial cells (HUVEC) were grown and lysed in hypotonic buffercontaining a cocktail of mammalian proteinase inhibitors. The celllysates were loaded on SDS-PAGE gels, transferred to nitrocellulosemembranes, blocked with dry fat-free milk in TBS, and overlaid with 0.5μg/ml of purified recombinant HagA. After three hours of incubation atambient temperature followed by washing, the membranes were treatedfirst with anti-HagA Mab and secondly with anti-mouse AP conjugate. TheHagA peptide was found to bind intensely to two proteins, ˜60 kDa and 65kDa in size, present in both epithelial and endothelial cells. The HagApeptide also bound to two heavy protein species, >200 kDa, present inendothelial cells. These results demonstrate that HagA binds to and intwith one or more proteins present in host cells and suggest the in vivoexistence of a protein complex between HagA and endothelial as well asepithelial proteins.

EXAMPLE 5

Proliferation Inhibition of HUH7 Cells by P. gingivalis Extracts, and byLive P. gingivalis Cells in the Presence/Absence of Inhibitors

Freshly collected whole P. gingivalis cells were used for 20-hourtreatment at a density of 2×10¹⁰ bacteria per ml DMEM (antibiotic-free).FIG. 11 represents the proliferation inhibition of HUH7 cells by P.gingivalis and E. coli extracts, and by live P. gingivalis cells in thepresence/absence of inhibitors (five stars: all cells remain attached,no proliferation inhibition). L-Cysteine was always present atconcentration of 5 mM to stabilize the anti-angiogenic activity. Theproliferation inhibition property of P. gingivalis extract and wholecells is clearly demonstrated on human hepatoma cell line (HUH7).

EXAMPLE 6

Immunofluorescent Microscopy of HUH7 Human Hepatoma Cells Treated withP. gingivalis Extract

Tissue culture cells grown in T-75 flasks at 37 C in DMEM (Pen/Strep) ina CO₂ incubator were subjected to 8 ml of trypsin-EDTA and incubated at37 C for 10-15 minutes for detachment. Trypsinized cells weretransferred (with 2×10 ml DMEM) to 50 ml culture tube and centrifuged at1K rpm for 10 seconds. Supernatant was removed and cells were washed at8 ml Ca-free PBS. Washed cells were centrifuged at K rpm for 10 secondsand excess wash was removed. Cells were resuspended in 20 ml of DMEM(Pen/Strep) media and transferred to new T-75 flask for incubation at 37C in 5% CO. HUH7 cells were incubated with P. gingivalis extract (0.8 mgprotein per ml of medium) for 20 h at which time the cells were washedthree times with phosphate buffered saline (PBS) and then fixed in 4%paraformaldehyde in PBS for 30 minutes at room temperature. This wasfollowed by washing twice in PBS and quenching in NH₄Cl (50 mM)/0.3%Tween 20/PBS for 10 minutes at room temperature. After quenching, theHUH7 were washed two times in PBS. The primary antibodies were rabbitanti-human occluding (Zymed Laboratories #71-1500) and rabbit anti-humanpan cadherin (Sigma Chemical Co., St Louis, Mo. #C3678). They werediluted 1/50 in PBS/5% normal goat serum/0.3% Tween™ 20 and applied tothe cells for 2 h at room temperature. The HUH7 were then washed fourtimes in PBS for 5 minutes each time. The secondary antibody(rhodamine-conjugated goat anti-rabbit (Sigma)) was applied for 1 h atroom temperature. The HUH7 cells were then washed twice with PBS beforemounting with Fluoromount-G (Southern Biotechnology Associates, Inc.,Birmingham Ala.) onto glass microscope slides and sealing with nailpolish. Images were viewed using an Olympus IX70 deconvolutionmicroscope and Delatvision software (Applied Precision, Inc., Wepahah,Wash.).

EXAMPLE 7

Reactivity with Anti-Occludin Antibody

Occludin-stained junctions degraded upon treatment with P. gingivalisextract. TLCK presence or heat-inactivation of P. gingivalis extractabolishes the activity. E. coli extract control treatment exhibited noactivity and the occludin network was intact. FIG. 12A shows non-treatedHUH7 cells. FIG. 12B shows HUH7 cells after treatment with P. gingivalisextract wherein the occludin network was degraded; FIG. 12C shows HUH7cells after treatment with P. gingivalis extract in the presence ofinhibitor TLCK, wherein the occludin network was intact; FIG. 12D showsHUH7 cells following treatment with heat-treated P. gingivalis extract,wherein the occludin network was degraded; and FIG. 12E shows HUH7 cellstreated with E. coli extract and demonstrates that E. coli does noteffect the occludin network. The data from immunoflourescent staining orjunctional molecules from HUH7 cells confirms the capacity of P.gingivalis extract to disrupt the intracellular network by degrading theborder consisting of cell adhesion molecules (CAMs).

EXAMPLE 8

Reactive with Anti-Pan Cadherin Antibody

Cadherin-stained junctions were degraded upon treatment with P.gingivalis extract. TLCK presence or heat-inactivation of P. gingivalisabolish the activity. FIG. 13A shows control, non-treated HUH7 hepatomacells, wherein the junctions were intact FIG. 13B shows HUH7 cellsfollowing treatment with P. gingivalis extract, wherein the junctionswere degraded; FIG. 13C shows HUH7 cells after treatment with P.gingivalis extract in the presence of inhibitor TLCK, wherein thejunctions were intact; and FIG. 13D shows HUH7 cells after treatmentwith heat-inactivated P. gingivalis extract, wherein the junction wereintact. The proliferation as stated above, and the data fromimmunoflourescent staining of junctional molecules from HUH7 cellsconfirms the capacity of P. gingivalis extract to disrupt theintracellular network by degrading the border consisting of celladhesion molecules. Thus both examples 7 and 8 demodulate that theunique activity of the present disclosed extract can be utilized fortumor disintegration.

EXAMPLE 9

Fractioning of Bacterial Culture Liquor Proteins

In order to partially purify the CAM-degrading activity, fractionalprecipitation of secreted P. gingivalis proteins from spent cultureliquor was achieved using ammonium sulfate. Broth culture grown in ananaerobic chamber was centrifuged for 20 minutes at a speed of 8000 rpm.Next it was filtered with a 0.2 μm filter (Nalgene) to remove anyremaining cells. To saturate to 60%, 36.1 g of (NH₄)₂SO₄ were dissolvedin every 100 ml of culture liquor. The solution was left stirringovernight at 4 C and collected the next day by centrifuging for 20minutes at 8000 rpm. Precipitated proteins were collected from sixliters of P. gingivalis W83 spent culture medium. The protein pellet wasresuspended in a 20 ml solution of 50 mM Tris HCl (pH 7.5). The solutionwas dialyzed (Pierce SnakeSkin tubing, 7 kDa MWCO) against 50 mMTris.HCl overnight at 4 C The dialysis was repeated with fresh buffer.Dialyzed solution was filtered with an Acrodisc® syringe filter (0.2 μm)and then concentrated using a Centriprep 10 (Amicon) for a total of anhour and a half After concentration, the solution was aliquoted, and theprotein concentration was determined using the BCA assay (Sigma B-9643).The solution was then stored at −80 C

EXAMPLE 10

Proliferation Inhibition of HUVEC Polarized Cell Line. HUVEC #Ecv-304Treatment with P. gingivalis Fractions

Polarized endothelial cells cultured on porous membrane inserts(Transwell, Corning Costar Corp., Cambridge, Mass.) were used as an invitro model for studying anti-angiogenic activities and to test fordifferential activity from both sides of the endothelium. Six hundred id(for 24-well plate) or 2.6 ml (for 6-well plate) of DMEM medium(Penn/Strep) were added to the lower chamber of tissue culture plates.Vascular endothelial cells were seeded into Corning Costar Transwellinserts in volumes of 0.1 ml medium (24-well plate) or 1.5 ml medium(6-well plate) in the upper chamber. Cultures were grown to confluencein a CO₂ incubator at 37.0 C before being treat The proteins were addedto the upper or lower chambers at a final concentration of 0.8 mg/ml.The cultures were incubated for 4 days in a CO₂ incubator. Similar retswere obtained using whole P. gingivalis cells (data not shown). FIG. 14Ashows control, untreated polarized human endothelial cells ECV-304; FIG.14B shows polarized ECV-304 cells treated basolaterally with 60%fraction of P. gingivalis culture liquid proteins; and FIG. 14C showspolarized ECV-304 cells following treatment lumenally with 60% fractionof P. gingivalis culture liquid proteins.

In each experiment the polarized endothelial cell layer was treated fromeither the apical or basolateral side with identical concentrations ofprotein preparations. As seen in the optical micrographs in FIGS. 14A, Band C at a point where complete destruction was observed frombasolateral application of ammonium sulfate-precipitated proteins, nodamage was observed in the cultures with lumenal (apical) application ofsame preparations. These data strongly support the conclusions thatanti-angiogenic activity is partially purified from P. gingivalissecreted proteins as 60-% fraction of ammonium sulfate-precipitatedculture liquor proteins; and the targeting of this activity toward thebasolateral, extravascular side of the vasculature is specificallybeneficial for degradation of the endothelial vascular cell layer inabnormally leaky tumor vessels. In addition to anti-angiogenic activity,immunofluorescent and proliferation inhibition studies with human cancercell lines (hepatoma and lung carcinoma) demonstrate the utility of thisP. gingivalis-associated activity for disintegration of extravasculartumor tissues, i.e., direct tumor-disintegration activity exists. Usingthe same abnormal openings to access both the basolateral side of thetumor vasculature and the surrounding tumor tissue brings double benefitto the proposed treatment.

In light of foregoing evidence, it is apparent that P. gingivalispolypeptides of the invention can be utilized as a vascular endothelialcell migration inhibitor and as an anti-angiogenic pharmaceutical agentFurthermore, while at present there does not appear to be any knowntherapeutic protocol based on selective degradation of cell-cell andcell-matrix adhesion molecules in tumors and a large number of otherdiseases, the present invention provides a new method of diseasetreatment of such pathologies. It is further predictable, based on thedisclosure provided herein, that other Porphyromonas gingivalis arginineor lysine specific cysteine protease polypeptides, hemagglutininpolypeptides and fragments thereof known or yet to be discovered thatexhibit similar anti-angiogenic activity, can be used according to themethods of this invention. Furthermore, combinations of such moleculescan also be used according to the methods of this invention

EXAMPLE 11

Effect of P. gingivalis on Integrin Receptors.

α5β1 integrin is upregulated in tumor endothelium (Stupack and Cheresh,Sci STKE. 2002 Feb. 12;2002(119):PE7). Being an apoptosis regulator, theintegrin is a target for anticancer drugs. B1 integrin is a target of P.gingivalis proteolytic activity in canine epithelial cells (Katz, etal., Infect Immun. 2000 Mar. 68(3):1441-9). To test if β1 integrin istargeted in a human cell line, immunoanalysis of detached HUVEC wasperformed after treatment with the P. gingivalis extract to determinethe extent of degradation. Western blots of SDS-PAGE gels were probedusing anti-human β1 integrin MAb (BD Transduction Labs, cat. #MMS-496R). The results demonstrated that treating with the P. gingivalisextract (except if heat-inactivated) resulted in complete degradation ofthe 130-kDa integrin.

EXAMPLE 12

Effect of P. gingivalis Proteins on Transendothelial Resistance (TER) ofPolarized HUVE Cells

Junctional complexes between adjacent polarized endothelial cellsconstitute a permeability barrier between lumenal and basolateralcompartments. Disruption of the junctional complexes leads to increasedflow of solutes, including small ions that can be monitoredelectrophysiologically by reading the changes in the transendothelialresistance (TER). HUVE cells were grown onto Transwell inserts with 0.4μm pore size. The integrity of the HUVEC layer was decreased bytreatment with P. gingivalis SPF compared to mock-treated control SeeFIG. 17 (the data are mean values from triplicate experiments). Notably,an identical concentration of SPF applied to the BL (basolateral)compartment decreased TER faster than when applied to the L (lumenal)compartment thus confirming and extending the observations made withcanine epithelial cells. (Katz et al., Infect Immun. 2000 March68(3):1441-9.)

FIG. 18 shows TER data and micrographs for an extended period of time (8days). Similar HUVEC treatment as above was performed in triplicate.FIG. 19 shows the TER graph FIG. 19 shows contrast micrographs of thecell layers on day 8. FIG. 19A; L-treated; FIG. 19B, BL-treated and FIG.19C, mock-treated control. The difference in the TER level reflects thestatus of the endothelial layer as documented on the micrographs,totally non-existing (BL treatment) and virtually intact (L treatment;control). Serine proteinase (trypsin) treatment (0.05% in medium) doesnot provide such specificity. Using electrophysiology and microscopy onthe same cell cultures, these data again demonstrate the destructiveaction of P. gingivalis secreted proteins on polarized human endothelialcell model and their preference for the BL side of the cell layer.

EXAMPLE 13

Endothelial Degradation Following Treatment with 15-kDa P. gingivalisProtein Cloned in E. coli.

A 15-kDa internal fragment of a HagA repeat (described in Paramaesvaran,et al., 1998, J. Dental Res., 77:664) was expressed in E. coli. In thisexperiment, a confluent endothelial cell layer was treated with asoluble sonic extract from E. coli expressing 15-kDa subunit of theadhesive domain (of gingipains or HagA), 0.5 mg total protein/mL FIG. 20shows micrographs of endothelial cell layer. FIG. 20A shows HUVECtreated with control E. coli host extract, 0.5 mg protein/mL FIG. 20Cshows control mock-treated cells. FIG. 20B shows eminent with E. coliexpressing 15-kDa protein.

FIG. 21 shows proliferation inhibition of HUVEC treated with 15-kDa P.gingivalis protein cloned in E. coli. (E. coli 0.5, control E. coliprotein extract at 0.5 mg/ml. E. coli HA2 0.5, E. coli protein extractcontaining HA2, the 15-kDa polypeptide at 0.5 me). The results of thisexperiment demonstrate sharp reduction of cell numbers upon treatmentwith recombinant 15-kDa P. gingivalis protein.

EXAMPLE 14

P. gingivalis cysteine proteinases and hemagglutinins can mediatedetachment of cells from a substrate and inhibit proliferation in humanendothelial and carcinoma cells. Also, junctional molecule degradationand vascular network disintegration using bacterial extracts and wholecells has been demonstrated. Further, partially purified secretedjunctional molecules-targeting proteinase have specificity toward thebasolateral side of polarized human endothelial monolayer.

In addition to the endothelial cell experiments, experiments with humancancer cell lines demonstrate the utility of this P.gingivalis-associated activity for treatment of extravascular tumortissues, i.e. direct tumor disintegration in addition to anti-angiogenicactivity. This activity can be specifically beneficial for degradationof the endothelial vascular cell layer from the basolateral side in theabnormally leaky tumor vessels. See FIG. 22. Using same abnormalopenings to access both the basolateral side of the tumor vasculatureand the extravascular tumor tissue would bring double benefit to theproposed treatment.

FIG. 22 shows potential pathways for leakage from blood vessels intumors. Intercellular openings (arrows) between lining cells of a murinetumor vessel viewed by scanning electron microscopy. Figures A and Bshow multiple large intercellular openings (arrow, 22A) and threesmaller transcellular holes (arrows, 22B) in branched lining cells of atumor vessel. The intercellular openings are much larger than the holes.Region in box in 22A is shown at higher magnification in 22B.(Hashizume, et al., Am J Pathol. 2000 Apr. 156(4):1363-80.)

1. A composition for the treatment, prevention, or amelioration of anangioproliferitive condition comprising a pharmaceutically effectiveamount of a substantially purified Porphyromonas gingivalis argininespecific cysteine protease polypeptide, Porphyromonas gingivalis lysinespecific cysteine protease polypeptide, Porphyromonas gingivalishemagglutinin polypeptide, or a combination thereof, wherein thepolypeptide has anti-angiogenic activity, and a pharmaceuticallyacceptable excipient.
 2. The composition of claim. 1, wherein thepolypeptide is selected from the group consisting of rgpA, rgpB, kgp,hag, prtT and tla polypeptides.
 3. The composition of claim 2, whereinthe polypeptide has at least about 90% sequence identity with a rgpA,rgpB, kgp, hag, prtT or tla polypeptide and wherein the polypeptide hasanti-angiogenic activity.
 4. The composition of claim 2, wherein thepolypeptide is a biologically functional homolog, or isoform of a rgpA,rgpB, kgp, hag, prtT or tla polypeptide.
 5. The composition of claim 1,wherein the polypeptide is a fragment of a Porphyromonas gingivalisarginine specific cysteine protease polypeptide, Porphyromonasgingivalis lysine specific cysteine protease polypeptide, orPorphyromonas gingivalis hemagglutinin polypeptide, and wherein thefragment has anti-angiogenic activity.
 6. The composition of claim 5,wherein the fragment is HA2.
 7. The composition of claim 1, wherein theangioproliferative condition is carcinoma, sarcoma, melanoma, benigntumor, ocular retinopathy, retrolental fibroplasias, psoriasis,angiofibromas, endometriosis, hemangioma, rheumatoid arthris, OslerWebber Syndrome, myocardial angiogenesis, telangiectasia, hemophiliacjoints, wound granulation, intestinal adhesions, post-surgery adhesions,arteriosclerosis, scleroderma, hypertrophic scars, cat scratch disease,Helicobacter pylori ulcers, capillary proliferation withinatherosclerotic plaque, or a combination thereof.
 8. A method for thetreatment, prevention, or amelioration of an angioproliferativecondition comprising administering a pharmaceutically effective amountof a Porphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cysteine proteasepolypeptide, Porphyromonas gingivalis hemagglutinin polypeptide, or acombination thereof wherein the polypeptide has anti-angiogenicactivity, to a patient in need thereof whereby the angioproliferativecondition is treated, prevented or ameliorated.
 9. The method of claim8, wherein the polypeptide is selected from the group co-ng of rgpA,rgpB, kgp, hag, prtT and tla polypeptides.
 10. The method of claim 8,wherein the polypeptide has at least about 90% sequence identity with argpA, rgpB, kgp, hag, or tla polypeptide and wherein the polypeptide hasanti-angiogenic activity.
 11. The method of claim 8, wherein thepolypeptide is a fragment of a Porphyromonas gingivalis argininespecific cysteine protease polypeptide, Porphyromonas gingivalis lysinespecific cysteine protease polypeptide, or a Porphyromonas gingivalishemagglutinin polypeptide, ad wherein the fragment has anti-angiogenicactivity.
 12. The method of claim 11, wherein the polypeptide fragmentis HA2.
 13. The method of claim 8, wherein the angioproliferativecondition is carcinoma, sarcoma, melanoma, benign tumor, ocularretinopathy, retrolental fibroplasias, psoriasis, angiofibromas,endometriosis, hemangioma, rheumatoid arthritis, Osler Webber Syndrome,myocardial angiogenesis, telangiectasia, hemophiliac joints, woundgranulation, intestinal adhesions, post-surgery adhesions,atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease,Helicobacter pylori ulcers, capillary proliferation withinatherosclerotic plaque, or a combination thereof.
 14. The method ofclaim 8, wherein the method comprises contacting a vasculature supplyinga biological structure affected by the angioproliferative condition withthe polypeptide.
 15. The method of claim 14, wherein the polypeptide iscontacted with a basolateral surface of the vasculature.
 16. A methodfor potentiating effects of a chemotherapeutically effective agentcomprising administering to a patient: (a) a Porphyromonas gingivalisarginine specific cysteine protease polypeptide, Porphyromonasgingivalis lysine specific cysteine protease polypeptid, Porphyromonasgingivalis hemagglutinin polypeptide, or a combination thereof, whereinthe polypeptide disrupts cell-cell adhesion, cell-matrix adhesion, orboth; and (b) a chemotherapeutically effective agent, whereby effects ofa chemotherapeutically effective agent are potentiated.
 17. The methodof claim 16, wherein the polypeptide is selected from the groupconsisting of rgpA, rgpB, kgp, hag, prtT and tla polypeptides.
 18. Themethod of claim 17, wherein the polypeptide has at least about 900%sequence identity with a rgpA, rgpB, kgp, hag, or tla polypeptide, andwherein the polypeptide has anti-angiogenic activity.
 19. The method ofclaim 16, wherein the polypeptide is a fragment of Porphyromonasgingivalis arginine specific cystine protease polypeptide, Porphyromonasgingivalis lysine specific cysteine protease polypeptide, orPorphyromonas gingivalis hemagglutinin polypeptide, and wherein thefragment has proteinase activity.
 20. The composition of claim 19,wherein the polypeptide fragment is HA2.
 21. A method for preventing theformation of new vasculature required for implantation or sustenance ofa fertilized an ovum comprising: administering a Porphyromonasgingivalis arginine specific cysteine protease polypeptide,Porphyromonas gingivalis lysine specific cysteine protease polypeptide,Porphyromonas gingivalis hemagglutinin polypeptide, or a combinationthereof to a mammal, wherein the polypeptide has anti-angiogenicactivity, whereby the formation of new vasculature required forimplantation or sustenance of a fertilized mammalian ovum is prevented.22. The method of claim 21, wherein the polypeptide is selected from thegroup consisting of rgpA, rgpB, kgp, hag, prtT and tla polypeptides. 23.The method of claim 22, wherein the polypeptide has at least about 90%sequence identity with a rgpA, rgpB, kgp, hag, prtT, or ta polypeptideand wherein the polypeptide has anti-angiogenic activity.
 24. The methodof claim 21, wherein the polypeptide is a fragment of a Porphyromonasgingivalis arginine specific cysteine protease polypeptide,Porphyromonas gingivalis lysine specific cysteine protease polypeptide,or Porphyromonas gingivalis hemagglutinin polypeptide, and wherein thefragment has anti-angiogenic activity.
 25. The method of claim 24,wherein the polypeptide fragment is HA2.
 26. A pharmaceuticalcomposition for facilitating passage of a compound through a blood-brainbarrier comprising a pharmaceutically effective amount of asubstantially purified Porphyromonas gingivalis arginine specificcysteine protease polypeptide, Porphyromonas gingivalis lysine specificcysteine protease polypeptide, a Porphyromonas gingivalis hemagglutininpolypeptide, or a combination thereof, and a pharmaceutically acceptableexcipient.
 27. The pharmaceutical composition of claim 26 furtherincluding a compound to be passed through the blood-brain barrier.
 28. Amethod delivering a compound through a blood-brain barrier of a patientcomprising administering a pharmaceutically effective amount of aPorphymonas gingivalis arginine specific cysteine protease polypeptide,Porphyromonas gingivalis lysine specific cysteine protease polypeptide,Porphyromonas gingivalis hemagglutinin polypeptide, or a combinationthereof and the compound, whereby the compound is passed through theblood-brain barrier.
 29. The method of claim 28, wherein the polypeptideis selected from the group consisting of rgpA, rgpB, kgp, hag, prtT andtla polypeptides.
 30. The method of claim 29, wherein the polypeptidehas at least about 90% sequence identity with a rgpA, rgpB, kgp, hag,prtT or tla polypeptide and wherein the polypeptide has anti-angiogenicactivity.
 31. The method of claim 28, wherein the polypeptide is afoment of a Porphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cysteine proteasepolypeptide, or a Porphyromonas gingivalis hemagglutinin polypeptide,and wherein the fragment has anti-angiogenic activity.
 32. The method ofclaim 31, wherein the polypeptide fragment is HA2.
 33. A method ofdegrading a tumor comprising: administering a pharmaceutically effectiveamount of a Porphyromonas gingivalis arginine specific cysteine proteasepolypeptide, Porphyromonas gingivalis lysine specific cysteine proteasepolypeptide, Porphyromonas gingivalis hemagglutinin polypeptide, or acombination thereof to a patient having a tumor, wherein the polypeptidedegrades cell-cell bonds, cell-matrix bonds, or both cell-cell bonds andcell-matrix bonds of the tumor, whereby the tumor is degraded.
 34. Themethod of claim 33 wherein the polypeptide is selected from the groupconsisting of rgpA, rgpB, kgp, hag, prtT and tla polypeptides.
 35. Themethod of claim 34 wherein the polypeptide has at least about 90% se ceidentity with a rgpA, rgpB, kgp, hag, prtT or tla polypeptide andwherein the polypeptide has anti-angiogenic activity.
 36. The method ofclaim 33 wherein the polypeptide is a fragment of a Porphyromonasgingivalis arginine specific cysteine protease polypeptide,Porphyromonas gingivalis lysine specific cysteine protease polypeptide,or Porphyromonas gingivalis is hemagglutinin polypeptide, and whereinthe fragment has anti-angiogenic activity.
 37. The method of claim 36wherein the polypeptide fragment is HA2.